Mitochondria and human immunodefiency virus type 1 transmission

ABSTRACT

Methods described herein relate to mitochondria and their role in Human immunodeficiency virus type 1 (HIV-1) infection and cell-to-cell HIV-1 transmission and compositions and methods for modulating mitochondrial mediated cell-to-cell transmission of HIV-1. Methods for screening to identify inhibitors of mitochondrial mediated cell-to-cell transmission of HIV-1 are also envisioned herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119(e) from U.SProvisional Application Ser. No. 61/547,925, filed Oct. 17, 2011, whichapplication is herein specifically incorporated by reference in itsentirety.

FIELD OF INVENTION

The present invention generally relates to the technical fields ofvirology and cellular biology. More particularly, the methods describedherein relate to mitochondria and their role in Human immunodeficiencyvirus type 1 (HIV-1) infection and transmission and compositions andmethods for modulating mitochondrial mediated infection and transmissionof HIV-1.

BACKGROUND OF INVENTION

AIDS (acquired immunodeficiency syndrome) is one of the leading causesof death in the developing world. HIV-1, a retrovirus which is a memberof the lentivirus subfamily, is the etiologic agent of AIDS. TheLentiviridae include non-oncogenic retroviruses which usually infectcells of the immune system, particularly macrophages and T cells,causing persistent infections in diseases with long incubation periodsand cytopathic effects in infected cells, such as syncytia and celldeath. Lentiviral infections are not cleared by the immune system, andlead to accumulated immunologic damage over a period of many years.

HIV-1 comprises an RNA genome and exhibits reverse transcriptaseactivity. During its growth cycle, HIV-1 copies its RNA into proviralDNA, which is able to integrate into the chromosomal DNA of the hostcell (provirus). Due to its retroviral nature and the small size of itsgenome, HIV-1 replication is strongly dependent on the host's cellmachinery. Thus, HIV uses the transcriptional and translationalmachinery of the host to express viral RNA and proteins and ultimatelyto release mature viruses from the cell by budding from the cytoplasmicmembrane. Viral replication of HIV-1 results in the death of host'shelper T cells, which leads to a state of severe immunodeficiency(AIDS), to the development of various malignancies and opportunisticinfections, and ultimately to the death of the infected organism.

HIV-1 is capable of infecting human host cells both through free viralparticles and through cell-to-cell transmission [1, 2]. Cell-to-cellspread is up to 10,000 times more efficient than free viral infection,because virus is shielded from cellular and immunological barriers [3,4, 5]. Several distinct modes of cell-to-cell HIV-1 dissemination havebeen reported, including virological synapses (VS) [6, 7], syncytia [8],filopodial bridges [9], and nanotubes [10].

Accordingly, the results presented herein elucidate certain of themechanisms involved in cell-to-cell transmission of HIV-1.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

Several publications and patent documents are referenced in thisapplication in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of thesepublications and documents is incorporated by reference herein.

SUMMARY OF THE INVENTION

The present inventors used live-cell, real-time fluorescence imaging ofco-cultures of HIV-1 infected T cells and uninfected target cells toexamine the action of mitochondria during cell-to-cell transmission ofthe virus. As described herein, mitochondria of HIV-1 infected cellsenter uninfected target cells and advance viral spread. The presentinventors, moreover, show that human mitochondria serve as viralreservoirs and carriers that can move between cells. Results presentedherein also show that purified mitochondria from HIV-1 infected cellsare infectious, and that mitochondrial inhibitors block HIV-1transmission. Viral infection and replication in the target cells wereverified by syncytial formation and HIV-1 core protein p24 production.An appreciation of the contribution of mitochondria to these processesoffers new insights into the cellular mechanisms of viral transmissionand identifies mitochondria as novel host targets for viral infection.

In accordance with the present discoveries, a method for screening toidentify a compound for treating a subject infected with humanimmunodeficiency virus type 1 (HIV-1) is presented, the methodcomprising contacting HIV-1 infected cells and target cells with acandidate compound, wherein the target cells are susceptible to HIV-1infection; and measuring cell-to-cell transmission of HIV-1 in thepresence and absence of the candidate compound, wherein a reduction incell-to-cell transmission of HIV-1 in the presence of the candidatecompound relative to the absence of the candidate compound indicatesthat the candidate compound or agent is a therapeutic agent for treatingthe subject infected with HIV-1.

In a particular embodiment, the candidate compound is a mitochondrialinhibitor or a compound suspected to exhibit properties of amitochondrial inhibitor. In a further embodiment thereof, themitochondrial inhibitor is ME-344. In a more particular embodiment, thecandidate compound is a mitochondrial inhibitor and a proteaseinhibitor. An exemplary compound exhibiting mitochondrial and proteaseinhibitor activity is bortezomib.

In another embodiment, the candidate compound is a modulator ofcytoskeletal processes or is a compound suspected to exhibit suchproperties. Such cytoskeletal processes include those relating to orassociated with actin or microtubule polymerization or depolymerization.

As described herein, cell-to-cell transmission of HIV-1 may be measuredby tracking mitochondrial movement from HIV-1 infected cells to thetarget cells. Measuring cell-to-cell transmission of HIV-1 may furthercomprise an assessment of virological synapse and syncytia formation.

In a further aspect, a method for screening to identify a compound thatinhibits human immunodeficiency virus type 1 (HIV-1) uptake bymitochondria is presented, the method comprising contacting HIV-1infected cells and target cells with a candidate compound, wherein thetarget cells are susceptible to HIV-1 infection; and measuring uptake ofHIV-1 by mitochondria in the target cells in the presence and absence ofthe candidate compound, wherein a reduction in uptake of HIV-1 bymitochondria in the target cells in the presence of the candidatecompound relative to the absence of the candidate compound indicatesthat the candidate compound is an inhibitor of HIV-1 uptake by themitochondria.

In an embodiment of the method, the candidate compound inhibits viralfusion or is a compound suspected to exhibit such properties. Exemplarysuch compounds include enfuvirtide (Fuzeon) and maraviroc (Selzentry).In another embodiment, the candidate compound inhibits activity of viralfusion protein gp41 or is a compound suspected to exhibit suchproperties. In yet another embodiment, the candidate compound inhibitsactivity of mitochondrial fusion proteins or is a compound suspected toexhibit such properties.

As described herein, measuring uptake of HIV-1 by mitochondria in thetarget cells may be determined by isolating mitochondria from the targetcells following contact with HIV-1 infected cells in the presence orabsence of the candidate compound and determining and comparinginfectivity of mitochondria isolated from target cells contacted withthe candidate compound to infectivity of mitochondria isolated fromtarget cells not contacted with the candidate compound, whereindecreased infectivity in the mitochondria isolated from target cellscontacted with the candidate compound indicates that the candidatecompound is an inhibitor of HIV-1 uptake by the mitochondria.

Also encompassed herein is a method for identifying a compound withanti-HIV-1 activity, the method comprising contacting HIV-1 infectedcells and target cells with a candidate compound, wherein the targetcells are susceptible to HIV-1 infection; and measuring cell-to-celltransmission of HIV-1 in the presence and absence of the candidatecompound, wherein a reduction in cell-to-cell transmission of HIV-1 inthe presence of the candidate compound relative to the absence of thecandidate compound identifies the candidate compound as a compound withanti-HIV-1 activity. In a particular embodiment, the candidate compoundbelongs to a class of mitochondrial inhibitors.

Also encompassed herein is a method for treating a subject infected withhuman immunodeficiency virus type 1 (HIV-1), the method comprisingadministering to the subject a mitochondrial inhibitor in atherapeutically effective amount, wherein the therapeutically effectiveamount is sufficient to reduce or inhibit cell-to-cell mediatedtransmission of HIV-1, thereby treating the subject infected HIV-1. In aparticular embodiment thereof, the mitochondrial inhibitor is ME-344,carbonyl cyanide m-chlorophenyl hydrazone (CCCP), antimycin A, oroligomycin. In another embodiment, the mitochondrial inhibitor does notexhibit the ability to inhibit nucleoside or nucleotide transcriptaseactivity. In yet another embodiment, the mitochondrial inhibitor is alsoa proteosome inhibitor. An exemplary such mitochondrial and proteosomeinhibitor is bortezomib.

In a further aspect, the method may further comprise treating thesubject with a therapeutic agent used for treating subjects infectedwith HIV-1, including, without limitation, nucleoside analogue reversetranscriptase inhibitors and either of a protease inhibitor or anon-nucleoside reverse transcriptase inhibitor.

In a further aspect, a method for reducing cell-to-cell mediatedtransmission of HIV-1 in a subject infected with human immunodeficiencyvirus type 1 (HIV-1) is presented, the method comprising administeringto the subject a mitochondrial inhibitor in a therapeutically effectiveamount, wherein the therapeutically effective amount is sufficient toreduce or inhibit cell-to-cell mediated transmission of HIV-1, therebyreducing cell-to-cell mediated transmission of HIV-1 in the subjectinfected HIV-1. In a particular embodiment thereof, the mitochondrialinhibitor is ME-344, carbonyl cyanide m-chlorophenyl hydrazone (CCCP),antimycin A, or oligomycin. In another embodiment, the mitochondrialinhibitor does not exhibit the ability to inhibit nucleoside ornucleotide transcriptase activity. In yet another embodiment, themitochondrial inhibitor is also a proteosome inhibitor. An exemplarysuch mitochondrial and proteosome inhibitor is bortezomib. The methodmay further comprise treating the subject with a therapeutic agent usedfor treating subjects infected with HIV-1, including, withoutlimitation, nucleoside analogue reverse transcriptase inhibitors andeither of a protease inhibitor or a non-nucleoside reverse transcriptaseinhibitor.

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing detailed description, whichproceeds with reference to the following illustrative drawings, and theattendant claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A (A-1 to A-5) and B (B-1 to B-5) shows live-cell time lapseimages of an HIV-1 infected H9 cell (upper) interacting with anuninfected GFP-MT2 target cell (lower), in co-culture. MitoTracker RedCMXRos stained mitochondria from the HIV-infected H9 cell are seen toenter the green GFP-MT2 target cell. A: A-1 to A-5; Blue, Red, and Greenoverlay. B: B-1 to B-5; Blue and Red overlay. Size bars 10 μm.

FIGS. 2A, B, C (C-1 to C-4), and D (D-1 to D-4) shows live-cellreal-time images of co-culture of HIV-1 infected H9 cells and uninfectedGFP-MT2 target cells.

-   A: One hour post-mixing, showing infected H9 cells with red    mitochondria and blue DNA, uninfected GFP-MT2 target cells in green,    and newly infected target cells with yellow staining (MitoTracker    Red on green). Various cellular structures, including nanotubes (NT,    green arrow), filopodia, and infectious cell centers (ICCs)    containing VS are seen around a large central syncytium (125×115    μm).-   B: At hour 20, most of the target cells have been depleted. The    original central syncytium has enlarged through the incorporation of    the surrounding target cells and ICCs. Three new satellite syncytia    are formed. MitoTracker Red stained red mitochondria increased    significantly within the original syncytium. Release of cell-free    mitochondria in culture is seen.-   C: C-1 to C-4; Transmission of red mitochondria (white arrows) from    infected cells in an ICC via a nanotube (NT, green arrow) to the    central syncytium, seen in a close up magnification of the area    outlined by white dotted lines in 2A. Selected time lapse images are    shown. Mitochondria migrate along the nanotubes at a rate of about    0.3 μm per minute.-   D: D-1 to D-4; Mitochondrial array: a new mode of mitochondrial    movement and transfer. At hour 11, a mitochondrial array (MA) of    MitoTracker Red—stained cell-free mitochondria emerges from one of    the HIV-infected cells (blue arrow). Over a 30 minute period, this    MA changed direction and navigated toward the syncytium. Selected    time-lapse images from Movie 1 are shown.-   Size bars in 2A and 2B, 25 μm, and in 2C and 2D, 10 μm.

FIGS. 3A, B and C (C-1 to C-12) shows a typical field of GFP-MT2 targetcells cultured in the presence of cell-free mitochondria purified fromHIV-infected H9 cells.

-   A: One hour after mixing: green target cells are all clustered into    ICC. Each ICC is attached to a tail-like structure made up of target    cells infected with cell-free mitochondria.-   B: The same field at hour 20: all target cells are infected and most    of the green target cells are depleted. Release of cell-free    mitochondria is seen.-   C: C-1 to C-12; Close up time-lapse images of white dotted area in    FIG. 3A: at hour one, entry of cell-free mitochondria into an MT2    target cell results in accumulation of mitochondria (yellow-orange)    at the site of entry. At hour 5 and 30 minutes, GFP expression    diminishes in the target cell, and the newly infected cell shows red    mitochondria and blue nuclear DNA.-   Size bars in 3A and 3B, 25 μm, and in 3C, 10 μm.

FIGS. 4A, B and C (C-1 to C-4) shows quantitation of HIV-1 spread bysyncytia formation and p24 expression.

-   A: Syncytial formation. HIV-1 induced syncytia formation was    measured in co-cultures of uninfected MT2 target cells with 1)    uninfected H9 cells, 2) mitochondria purified from uninfected H9    cells, 3) HIV-1 infected H9 cells, 4) mitochondria purified from    HIV-1 infected H9 cells, and 5) HIV-1 infected H9 cells treated with    the mitochondria inhibitor CCCP. Syncytia formation was expressed as    number per well. No syncytia were detected with uninfected H9 cells,    or with purified cell-free mitochondria from uninfected cells.    Syncytial formation was seen in co-cultures of MT2 cells with    HIV-infected H9 cells, and with cell-free mitochondria purified from    HIV-infected H9 cells. Treatment of HIV-infected H9 cells with 10 μM    CCCP for 3 hours inhibited syncytia formation by 95%.-   B: HIV-1 antigen p24 production. Viral antigen p24 production was    measured in the supernatants of co-cultures as described above and    expressed as ng/ml.-   C: C-1 to C-4; Live-cell fluorescent images of syncytial formation.    GFP-MT2 target cells are green. H9 uninfected cells and HIV-1    infected H9 cells were fluorescently labeled with MitoTracker Red    CMXRos to stain mitochondria red and Hoechst 33342 to stain DNA    blue. Images shown are co-cultures of uninfected MT2 target cells    with HIV-1 infected H9 cells (4C-1), uninfected H9 cells (4C-2),    HIV-1 infected H9 cells treated with CCCP (4C-3), and mitochondria    purified from HIV-1 infected H9 cells (4C-4). Syncytial formation    was detected in co-cultures of MT2 cells with HIV-infected H9 cells,    and with cell-free mitochondria purified from HIV-infected H9 cells.    Treatment of HIV-infected H9 cells with CCCP blocked syncytia    formation. No syncytia were detected with uninfected H9 cells, or    with purified cell-free mitochondria from uninfected cells (not    shown). Size bars 25 μm.

DETAILED DESCRIPTION OF THE INVENTION

HIV-1 is known to be disseminated via several distinct modes, includingvirological synapses (VS) [6, 7], syncytia [8], filopodial bridges [9],and nanotubes [10]. The present inventors hypothesized that formation ofthese structures would require metabolic energy and thus involvemitochondria. The precise roles of mitochondria during cell-to-cellspread of HIV-1 are unknown. As described herein, the present inventorsused live-cell, real-time fluorescence imaging of co-cultures of HIV-1infected T cells and uninfected target cells to examine the action ofmitochondria during viral transmission. Findings presented hereindemonstrate that mitochondria of HIV-1 infected cells enter uninfectedtarget cells and promote cell-to-cell viral transmission. Moreover,cell-free mitochondria purified from HIV-infected cells are infectiousand mitochondrial inhibitors inhibit cell-to-cell transmission of HIV-1.These findings show that mitochondria of HIV-1 infected cells playindispensible roles in cell-to-cell transmission of HIV and serve asviral reservoirs and carriers. Results presented herein thus offer newinsights into the cellular mechanisms of viral transmission and suggestthat mitochondria may be new targets for therapeutic intervention. Thetransfer of organelles between cells is a novel finding that has notbeen previously reported. Cell-to-cell transfer of mitochondria may be ageneral phenomenon, and its role in viral transmission may be only oneof many implications. The role of mitochondria as a viral reservoir hasimplications for viral persistence and shielding from host immuneresponses.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A. Terminology

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, reference to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

“Pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopoeia orother generally recognized pharmacopoeia for use in animals, and moreparticularly in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound of theinvention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. Such saltsinclude: (1) acid addition salts, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike. Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium, and the like; and whenthe compound contains a basic functionality, salts of non toxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like. The term“pharmaceutically acceptable cation” refers to a non toxic, acceptablecationic counter-ion of an acidic functional group. Such cations areexemplified by sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium cations, and the like.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with which a compound of the invention isadministered.

“Preventing” or “prevention” refers to a reduction in risk of acquiringa disease or disorder (i.e., causing at least one of the clinicalsymptoms of the disease not to develop in a subject that may be exposedto or predisposed to the disease but does not yet experience or displaysymptoms of the disease).

“Prodrugs” refers to compounds, including derivatives of the compoundsof the invention, which have cleavable groups and become by solvolysisor under physiological conditions the compounds of the invention whichare pharmaceutically active in vivo. Such examples include, but are notlimited to, choline ester derivatives and the like, N-alkylmorpholineesters and the like.

“Solvate” refers to forms of the compound that are associated with asolvent, usually by a solvolysis reaction. Conventional solvents includewater, ethanol, acetic acid and the like. The compounds of the inventionmay be prepared e.g. in crystalline form and may be solvated orhydrated. Suitable solvates include pharmaceutically acceptablesolvates, such as hydrates, and further include both stoichiometricsolvates and non-stoichiometric solvates.

“Subject” means any animal or artificially modified animal. Animalsinclude, but are not limited to, humans, non-human primates, cows,horses, sheep, goats, pigs, dogs, cats, rabbits, ferrets, rodents suchas mice, rats and guinea pigs, and birds and fowl, such as chickens andturkeys. Artificially modified animals include, but are not limited to,transgenic animals or SCID mice with human immune systems. In thepreferred embodiment, the subject is a human. Indeed, the terms “human,”“patient” and “subject” can be used interchangeably herein.

“Therapeutically effective amount” means the amount of a compound that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” can vary depending on the compound, the disease and itsseverity, and the age, weight, etc., of the subject to be treated.

“Treating” or “treatment” of any disease or disorder refers, in oneembodiment, to ameliorating the disease or disorder (i.e., arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In another embodiment “treating” or “treatment”refers to ameliorating at least one physical parameter, which may not bediscernible by the subject. In yet another embodiment, “treating” or“treatment” refers to modulating the disease or disorder, eitherphysically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.With respect HIV-1 infection, treatment may inhibit or decrease HIV-1infection or cell-to-cell transmission thereof.

As used herein, the term “operably linked” refers to a regulatorysequence capable of mediating the expression of a coding sequence andwhich is placed in a DNA molecule (e.g., an expression vector) in anappropriate position relative to the coding sequence so as to effectexpression of the coding sequence. This same definition is sometimesapplied to the arrangement of coding sequences and transcription controlelements (e.g. promoters, enhancers, and termination elements) in anexpression vector. This definition is also sometimes applied to thearrangement of nucleic acid sequences of a first and a second nucleicacid molecule wherein a hybrid nucleic acid molecule is generated.

A “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage orvirus, to which another genetic sequence or element (either DNA or RNA)may be attached so as to bring about the replication of the attachedsequence or element.

An “expression vector” or “expression operon” refers to a nucleic acidsegment that may possess transcriptional and translational controlsequences, such as promoters, enhancers, translational start signals(e.g., ATG or AUG codons), polyadenylation signals, terminators, and thelike, and which facilitate the expression of a polypeptide codingsequence in a host cell or organism.

The terms “transform”, “transfect”, or “transduce”, shall refer to anymethod or means by which a nucleic acid is introduced into a cell orhost organism and may be used interchangeably to convey the samemeaning. Such methods include, but are not limited to, transfection,electroporation, microinjection, PEG-fusion and the like.

The introduced nucleic acid may or may not be integrated (covalentlylinked) into nucleic acid of the recipient cell or organism. Inbacterial, yeast, plant and mammalian cells, for example, the introducednucleic acid may be maintained as an episomal element or independentreplicon such as a plasmid. Alternatively, the introduced nucleic acidmay become integrated into the nucleic acid of the recipient cell ororganism and be stably maintained in that cell or organism and furtherpassed on or inherited to progeny cells or organisms of the recipientcell or organism. In other applications, the introduced nucleic acid mayexist in the recipient cell or host organism only transiently.

The phrase “consisting essentially of” when referring to a particularnucleotide or amino acid means a sequence having the properties of agiven SEQ ID NO:. For example, when used in reference to an amino acidsequence, the phrase includes the sequence per se and molecularmodifications that would not affect the basic and novel characteristicsof the sequence.

The term “isolated” refers to the state in which specific bindingmembers of the invention, or nucleic acid encoding such binding memberswill be, in accordance with the present invention. Members and nucleicacid will be free or substantially free of material with which they arenaturally associated such as other polypeptides or nucleic acids withwhich they are found in their natural environment, or the environment inwhich they are prepared (e.g. cell culture) when such preparation is byrecombinant DNA technology practiced in vitro or in vivo. Members andnucleic acid may be formulated with diluents or adjuvants and still forpractical purposes be isolated—for example the members will normally bemixed with gelatin or other carriers if used to coat microtiter platesfor use in immunoassays, or will be mixed with pharmaceuticallyacceptable carriers or diluents when used in diagnosis or therapy.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to probes of thepresent invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

Primers may be labeled fluorescently with 6-carboxyfluorescein (6-FAM).Alternatively primers may be labeled with 4, 7, 2′,7′-Tetrachloro-6-carboxyfluorescein (TET). Other alternative DNAlabeling methods are known in the art and are contemplated to be withinthe scope of the invention.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight of a given material (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-95% by weightof the given compound. Purity is measured by methods appropriate for thegiven compound (e.g. chromatographic methods, agarose or polyacrylamidegel electrophoresis, HPLC analysis, and the like). “Mature protein” or“mature polypeptide” shall mean a polypeptide possessing the sequence ofthe polypeptide after any processing events that normally occur to thepolypeptide during the course of its genesis, such as proteolyticprocessing from a polypeptide precursor. In designating the sequence orboundaries of a mature protein, the first amino acid of the matureprotein sequence is designated as amino acid residue 1.

The term “tag”, “tag sequence” or “protein tag” refers to a chemicalmoiety, either a nucleotide, oligonucleotide, polynucleotide or an aminoacid, peptide or protein or other chemical, that when added to anothersequence, provides additional utility or confers useful properties tothe sequence, particularly with regard to methods relating to thedetection or isolation of the sequence. Thus, for example, a homopolymernucleic acid sequence or a nucleic acid sequence complementary to acapture oligonucleotide may be added to a primer or probe sequence tofacilitate the subsequent isolation of an extension product orhybridized product. In the case of protein tags, histidine residues(e.g., 4 to 8 consecutive histidine residues) may be added to either theamino- or carboxy-terminus of a protein to facilitate protein isolationby chelating metal chromatography. Alternatively, amino acid sequences,peptides, proteins or fusion partners representing epitopes or bindingdeterminants reactive with specific antibody molecules or othermolecules (e.g., flag epitope, c-myc epitope, transmembrane epitope ofthe influenza A virus hemaglutinin protein, protein A, cellulose bindingdomain, calmodulin binding protein, maltose binding protein, chitinbinding domain, glutathione S-transferase, and the like) may be added toproteins to facilitate protein isolation by procedures such as affinityor immunoaffinity chromatography. Chemical tag moieties include suchmolecules as biotin, which may be added to either nucleic acids orproteins and facilitates isolation or detection by interaction withavidin reagents, and the like. Numerous other tag moieties are known to,and can be envisioned by, the trained artisan, and are contemplated tobe within the scope of this definition.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5 ×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20° C. below the predicted or determined T_(M) with washes of higherstringency, if desired.

As used herein, an “agent”, “candidate compound”, or “test compound” maybe used to refer to, for example, nucleic acids (e.g., DNA and RNA),carbohydrates, lipids, proteins, peptides, peptidomimetics, smallmolecules and other drugs. In particular the term agent includescompounds such as test compounds or drug candidate compounds.

The term “control substance”, “control agent”, or “control compound” asused herein refers a molecule that is inert or has no activity relatingto an ability to modulate a biological activity. With respect to thepresent disclosure, such control substances are inert with respect to anability to modulate mitochondrial activity. Exemplary controls include,but are not limited to, solutions comprising physiological saltconcentrations.

The term ‘agonist’ refers to a ligand that stimulates the receptor theligand binds to in the broadest sense or stimulates a response thatwould be elicited on binding of a natural binder to a binding site.

The term ‘assay’ means any process used to measure a specific propertyof a compound or agent. A ‘screening assay’ means a process used tocharacterize or select compounds based upon their activity from acollection of compounds.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “1” means liter.

The compositions containing the molecules, compounds, or agentsdescribed herein can be administered for therapeutic purposes. Intherapeutic applications, compositions are administered to a patientalready suffering from an HIV-1 infection (such as, e.g., an AIDSpatient) in an amount sufficient to at least partially arrest thesymptoms of the disease and its complications. An amount adequate toaccomplish this is defined as a “therapeutically effective amount ordose.” Amounts effective for this use will depend on the severity of thedisease and the weight and general state of the patient.

“Infection”, as the term is used herein, generally relates to the entry,replication, insertion, lysis or other event or process involved in thepathogenesis of a virus with respect to a host cell. Thus, decreasinginfection includes decreasing entry, replication, insertion, lysis,cell-to-cell transmission, or other pathogenic process of a virus in acell or subject, or combinations thereof. Infection includes theintroduction of an infectious agent, such as a non-recombinant virus,recombinant virus, plasmid, or other agent capable of infecting a host,such as the cell of a subject.

“Exposed” to HIV-1 means contact or association with HIV-1 such thatinfection could result.

“HIV-1 infected” means the introduction of viral components, virusparticles, or viral genetic information into a cell, such as by fusionof the cell membrane with HIV-1. The cell may be a cell of a subject. Inthe preferred embodiment, the cell is a cell in a human subject.

B. Detailed Disclosure

The invention relates generally to methods and agents for inhibitingmitochondrial mediated transmission of HIV-1. Prior to the presentdiscoveries, the role of mitochondria in the cell-to-cell transmissionof HIV-1 was unappreciated. The discovery that mitochondria play acritical role in cell-to-cell transmission of HIV-1 provides for novelmethods for treating HIV-1 disease using mitochondrial inhibitors,either alone or in conjunction with known anti-HIV-1 therapeutic agentsand regimens, and assays for screening and identifying agents, compoundsor peptides to modulate mitochondrial activity that may be used toidentify novel therapeutics for treating AIDS patients.

Further to the above, the present inventors demonstrate herein thatfunctional mitochondria are essential for cell-to-cell transmission ofthe virus by, for example, pre-treating HIV-1 infected H9 cells withmitochondrial inhibitors including carbonyl cyanidem-chlorophenylhydrazone (CCCP, 10 μM), antimycin A (2.4 μM), andoligomycin (2.4 μM) for 3 hours and evaluating the effect of theseinhibitors on cell-to-cell viral HIV-1 transmission. The concentrationsused for the indicated inhibitors conferred maximum inhibition of thetargeted metabolic step, but caused no cell death. After pre-treatment,cells were washed to remove free drug, suspended in complete medium, andlabeled with MitoTracker Red and Hoechst 33342. Pre-treated cells werethen co-cultured with GFP-MT2 target cells, and cell-to-celltransmission of HIV-1 was analyzed. As shown in FIG. 4C-3, mitochondrialinhibitors reduced cell-to-cell spread of HIV-1 and decreased VS andsyncytia formation. Accordingly, mitochondrial inhibitors are usefulcompounds/agents for inhibiting HIV-1 cell-to-cell transmission. Inlight of these findings, methods are presented herein to screen knownmitochondrial inhibitors to evaluate their ability to inhibit viral(e.g., HIV-1) cell-to-cell transmission and to identify newmitochondrial inhibitors that can be used in methods to inhibit viral(e.g., HIV-1) cell-to-cell transmission.

In vivo animal models of HIV-1 or HIV-1-like viral infection or HIV-1immune response may be utilized by the skilled artisan to further oradditionally screen, assess, and/or verify mitochondrial inhibitors, oragents identified using screening methods of the present invention, asuseful for treating HIV-1 infected subjects in vivo. Such animal modelsinclude, but are not limited to models of immune system modulation orimmune response. In particular, HIV models, including “humanized” micemodels are known and can be utilized. Immunodeficient mice are engraftedwith a human immune system using various sources of hematopoietic stemcells, depending on the model (CD34+cells from fetal liver, from cordblood, etc). Humanized mice such as, for example, humanized bonemarrow-liver-thymus (BLT) mice (Wege A K et al 92008) Curr Top MicrobiolImmunol 324:149-165; Denton P W et al (2008) PLoS January 15; 5(1):e16)may be challenged with HIV-1 after immunization. DKO-hu HSC mice mayalso be used as a humanized mouse model susceptible to HIV infection(Zhang L et al (2007) Blood 109(7):2978-81). Hu-PBL-SCID mice have,moreover, been immunized with IFN-DCs and pulsed with inactivated HIV-1or infected with HIV-1 to assess response and protection (Lapenta C etal (2003) J Exp Med 198(2):361-7).

Mitochondrial inhibitors or agents identified using screening methods ofthe present invention may be administered to a patient in need oftreatment via any suitable route, including by intravenous,intraperitoneal, intramuscular injection, or orally.

Mitochondrial Inhibitors

Exemplary mitochondrial inhibitors of the invention, include, withoutlimitation: carbonyl cyanide m-chlorophenyl hydrazone (CCCP;H⁺ionophore), antimycin A and myxothiazol (inhibitors of complex III),oligomycin, and rotenone (inhibitor of complex I). CCCP is a chemicalinhibitor of oxidative phosphorylation. It is a nitrile, hydrazone andionophore. CCCP affects protein synthesis reactions in mitochondria.CCCP causes an uncoupling of the proton gradient that is establishedduring the normal activity of electron carriers in the electrontransport chain. CCCP behaves predominantly as an ionophore and reducesthe ability of ATP synthase to function optimally.

Antimycin A binds to the Qi site of cytochrome c reductase, therebyinhibiting the oxidation of ubiquinol in the electron transport chain ofoxidative phosphorylation. The inhibition of this reaction disrupts theformation of the proton gradient across the inner membrane. Theproduction of ATP is subsequently inhibited, as protons are unable toflow through the ATP synthase complex in the absence of a protongradient. This inhibition also results in the formation of quantities ofthe toxic free radical superoxide.

Oligomycin inhibits ATP synthase by blocking its proton channel, whichis necessary for oxidative phosphorylation of ADP to ATP (energyproduction). The inhibition of ATP synthesis also inhibits the electrontransport chain. Because the high proton concentration build up is notdissipated, the free energy released by biological oxidation ofsubstrates is not enough to pump any more protons against the steepgradient. A potential complication of administering oligomycin to anindividual is the accumulation of high levels of lactate in the bloodand urine. Such potentialities would have to be monitored diligently andmeasures taken to correct for elevated levels of lactate in anyindividual treated with oligomycin. Oligomycin is also an inhibitor ofATP synthase. In oxidative phosphorylation research, it is used toprevent state 3 (phosphorylating) respiration. Oligomycin also hasutility as an antibiotic.

Mitochondrial inhibitors, such as those proposed for the treatment ofcertain cancers are also encompassed herein. Such mitochondrialinhibitors include, without limitation, efrapeptin F, which is amitochondrial complex V inhibitor, and inhibitors of complex I, II, III,and other inhibitors of complex V. Such mitochondrial inhibitors showpreferential cytotoxicity to human pancreatic cancer PANC-1 cells underglucose-deprived conditions. See Momose et al. (Biochem. Biophys. Res.Comm. 392:460-466, 2010), the entire of contents of which isincorporated herein in its entirety. Mitochondrial inhibitors and usesthereof are, moreover, described in Galluzzi et al. (Oncogene25:4812-4830, 2006) and these inhibitors and guidance relating to theiruse is applicable to the treatment of patients afflicted with HIV-1. Theentire content of Galluzzi et al. (Oncogene 25:4812-4830, 2006) isincorporated herein in its entirety.

Also encompassed herein are the mitochondrial inhibitors NV-128 andME-344 and uses thereof for treating patients afflicted with HIV-1.NV-128, for example, has demonstrated activity against a broad range ofcancers I pre-clinical studies. NV-128 treatment of cancer cells inducesa rapid loss of cellular energy resulting in the inhibition of bothmammalian target of rapamycin (mTOR1 and mTOR2) pathways. Pre-clinicalstudies using NV-128 reveal that it induces mitochondrial instabilitythat ultimately leads to cell death in otherwise chemotherapy-resistantovarian cancer stem cells. See, for example, Alvero et al. Cancer 115:3204-3216, 2009; Alvero et al. Mol Cancer Ther 10:1385-1393, 2011.

ME-344 is an active metabolite of NV-128 that exhibits superioranti-tumor activity against a broad spectrum of human cancer cell linesrelative to that of NV-128 in pre-clinical studies. A Phase I clinicaltrial of intravenously administered ME-344 in patients with solidrefractory tumors is ongoing. The protocol calls for five escalatingdose cohorts to evaluate safety and tolerability of intravenous (iv)ME-344. The trial is also designed to characterize the pharmacokineticprofile of ME-344 and reveal any preliminary clinical anti-tumoractivity. Intravenous infusions of ME-344 are administered once weeklyfor three weeks and may optionally, continue with weekly dosing pendingsafety assessment and evidence of clinical benefit. With regard toME-344, the content of European Patent No. 1 794 141 is incorporatedherein by reference in its entirety.

The IUPAC/chemical name of ME-344 is4,4′-(7-hydroxy-8-methylchroman-3,4-diyl)diphenol. The chemicalstructure of ME-344 is as follows:

In another embodiment, the mitochondrial inhibitor is dichloroacetate(DCA), an inhibitor of mitochondrial pyruvate dehydrogenase kinase. DCAhas been evaluated in clinical trials for the treatment of malignantglioma and glioblastome multiforme. DCA is administered orally and is asmall molecule that readily crosses the blood brain barrier to accessHIV-1 infected cells in the brain. Additional information pertaining to,for example, dosing parameters used in the Phase II Clinical studydirected to the above can be found via the formal title: “A Phase IIOpen-labeled, Double-arm Clinical Study of Dichloroacetate (DCA) inMalignant Gliomas and Glioblastome (GMB) Patients” as presented inpublicly available web sites directed to clinical trials and provided asa service by the U.S. National Institutes of Health.

In a further embodiment, a mitochondrial inhibitor may also exhibitproteosome inhibitor activity. An exemplary such mitochondrial andproteosome inhibitor is bortezomib. This compound has been evaluated inPhase I, II, and II clinical trials. See, for example, Ling et al. (JBiol Chem 278:33714-33723, 2003) and references cited therein, theentire content of each of which is incorporated herein by reference.

Methods of Screening to Identify Inhibitors of Mitochondrial MediatedHIV-1 Transmission

Suitable screening methods are set forth in the Examples presentedherein. As described herein, a suitable source of HIV-1 virus, such asH9 cells infected with HIV-1, or an isolate of purified HIV-1(HIV-IIIB), and target cells that are susceptible to HIV-1 infection,such as MT2 cells, are co-cultured to achieve infection. Infected cellsand target cells can be differentially labeled using, for example, afluorescent label [e.g., MitoTracker Red CMXRos and green fluorescentprotein (GFP-MT2)] to differentiate the cellular origin of organellesvisualized in co-cultures thereof. Live-cell real-time images and timelapse movies can be used to visualize HIV-1 infection and transmission.Syncytia formation assays can also be used to assess acute HIV-1infection. Viral replication can also be assayed by measuring HIV-1 coreprotein p24 expression in the co-culture. HIV-1 core protein p24expression may be assayed using a variety of means includingcommercially available ELISA kits. Syncytia formation assays and HIV-1core protein p24 expression assays are described in detail in Lee-Huanget al., Structural and Functional Modeling of Human Lysozyme Reveals aUnique Nonapeptide, HL9, with Anti-HIV Activity. Biochemistry 44 (2005)4648-4655, the entire contents of which is incorporated herein in itsentirety.

Other suitable target cells, which are susceptible to HIV-1 infection,are known in the art and include, without limitation, any CD4 positive Tcells or other types of cells, including monocytes, primary cells, andbody fluids comprising target cells.

With regard to potential sources of HIV-1, any isolates and/or bodyfluids derived from AIDS patients or HIV-1 positive subjects, as well asother laboratory strains may be utilized.

Individual compounds or libraries of compounds can be screened using anyone or all of the above assays to determine if the presence of thecompound in the co-culture reduces mitochondrial mediated cell-to-celltransmission of HIV-1 relative to co-cultures incubated in the presenceof control compound.

Pharmaceutical Compositions

When employed as pharmaceuticals, mitochondrial inhibitors or agentsidentified using screening methods of the present invention aretypically administered in the form of a pharmaceutical composition. Suchcompositions can be prepared in a manner well known in thepharmaceutical art and comprise at least one active compound.

Generally, mitochondrial inhibitors or agents of this invention areadministered in a pharmaceutically effective amount. The amount of themitochondrial inhibitor or agent actually administered will typically bedetermined by a physician, in the light of the relevant circumstances,including the condition to be treated, the chosen route ofadministration, the actual compound -administered, the age, weight, andresponse of the individual patient, the severity of the patient'ssymptoms, and the like.

The mitochondrial inhibitors or agents identified using screeningmethods of the present invention may be used alone or in combinationwith other therapeutic agents used for treating subjects infected withHIV-1. Accordingly, pharmaceutical compositions may comprise one or moremitochondrial inhibitors or agents identified using screening methods ofthe present invention and such compositions may further comprise one ormore other therapeutic agents used for treating subjects infected withHIV-1. Such therapeutic agents include: nucleoside analogue reversetranscriptase inhibitors and either of protease inhibitors ornon-nucleoside reverse transcriptase inhibitors. For such a combinedtherapeutic approach, compositions may comprise all of the above ormultiple compositions may be formulated and administered to a subject inneed thereof, concomitantly or at appropriately space intervals foroptimal therapeutic efficacy.

Examples of nucleoside analogue reverse transcriptase inhibitors (NRTIs)include: Abacavir (Ziagen), and the combination of drugs emtricitabineand tenofovir (Truvada) and lamivudine and zidovudine (Combivir).Examples of protease inhibitors include: atazanavir (Revataz), darunavir(Prezista), fosamprenavir (Lexiva), and ritonavir (Norvir). Examples ofnon-nucleoside reverse transcriptase inhibitors (NNRTIs) include:efavirenz (Sustiva), etravirine (Intelence), and nevirapine (Viramune).

Other agents used for the treatment of subjects infected with HIV-1include entry or fusion inhibitors, including enfuvirtide (Fuzeon) andmaraviroc (Selzentry), and integrase inhibitors, such as raltegravir(Isentress).

The pharmaceutical compositions of this invention can be administered bya variety of routes including oral, rectal, transdermal, subcutaneous,intravenous, intramuscular, and intranasal. Depending on the intendedroute of delivery, the mitochondrial inhibitors or agents of thisinvention are preferably formulated as either injectable or oralcompositions or as salves, as lotions or as patches all for transdermaladministration.

The compositions for oral administration can take the form of bulkliquid solutions or suspensions, or bulk powders. More commonly,however, the compositions are presented in unit dosage forms tofacilitate accurate dosing. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient. Typical unitdosage forms include prefilled, premeasured ampules or syringes of theliquid compositions or pills, tablets, capsules or the like in the caseof solid compositions. In such compositions, the furansulfonic acidcompound is usually a minor component (from about 0.1 to about 50% byweight or preferably from about 1 to about 40% by weight) with theremainder being various vehicles or carriers and processing aids helpfulfor forming the desired dosing form.

Liquid forms suitable for oral administration may include a suitableaqueous or nonaqueous vehicle with buffers, suspending and dispensingagents, colorants, flavors and the like. Solid forms may include, forexample, any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based upon injectable sterilesaline or phosphate-buffered saline or other injectable carriers knownin the art. As before, the active compound in such compositions istypically a minor component, often being from about 0.05 to 10% byweight with the remainder being the injectable carrier and the like.

Transdermal compositions are typically formulated as a topical ointmentor cream containing the active ingredient(s), generally in an amountranging from about 0.01 to about 20% by weight, preferably from about0.1 to about 20% by weight, preferably from about 0.1 to about 10% byweight, and more preferably from about 0.5 to about 15% by weight. Whenformulated as an ointment, the active ingredients will typically becombined with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream with,for example an oil-in-water cream base. Such transdermal formulationsare well-known in the art and generally include additional ingredientsto enhance the dermal penetration of stability of the active ingredientsor the formulation. All such known transdermal formulations andingredients are included within the scope of this invention.

The compounds of this invention can also be administered by atransdermal device. Accordingly, transdermal administration can beaccomplished using a patch either of the reservoir or porous membranetype, or of a solid matrix variety.

The above-described components for orally administrable, injectable ortopically administrable compositions are merely representative. Othermaterials as well as processing techniques and the like are set forth inPart 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, MackPublishing Company, Easton, Pennsylvania, which is incorporated hereinby reference.

The mitochondrial inhibitors or agents of this invention (i.e.,compounds) can also be administered in sustained release forms or fromsustained release drug delivery systems. A description of representativesustained release materials can be found in Remington's PharmaceuticalSciences.

The following formulation examples illustrate representativepharmaceutical compositions of this invention. The present invention,however, is not limited to the following pharmaceutical compositions.

Formulation 1—Tablets

A compound of the invention is admixed as a dry powder with a drygelatin binder in an approximate 1:2 weight ratio. A minor amount ofmagnesium stearate is added as a lubricant. The mixture is formed into240-270 mg tablets (80-90 mg of active amide compound per tablet) in atablet press.

Formulation 2—Capsules

A compound of the invention is admixed as a dry powder with a starchdiluent in an approximate 1:1 weight ratio. The mixture is filled into250 mg capsules (125 mg of active amide compound per capsule).

Formulation 3—Liquid

A compound of the invention (125 mg), sucrose (1.75 g) and xanthan gum(4 mg) are blended, passed through a No. 10 mesh U.S. sieve, and thenmixed with a previously made solution of microcrystalline cellulose andsodium carboxymethyl cellulose (11:89, 50 mg) in water. Sodium benzoate(10 mg), flavor, and color are diluted with water and added withstirring. Sufficient water is then added to produce a total volume of 5mL.

Formulation 4—Tablets

A compound of the invention is admixed as a dry powder with a drygelatin binder in an approximate 1:2 weight ratio. A minor amount ofmagnesium stearate is added as a lubricant. The mixture is formed into450-900 mg tablets (150-300 mg of active amide compound) in a tabletpress.

Formulation 5—Injection

A compound of the invention is dissolved or suspended in a bufferedsterile saline injectable aqueous medium to a concentration ofapproximately 5 mg/ml.

Formulation 6—Topical

Stearyl alcohol (250 g) and a white petrolatum (250 g) are melted atabout 75° C. and then a mixture of a compound of the invention (50 g)methylparaben (0.25 g), propylparaben (0.15 g), sodium lauryl sulfate(10 g), and propylene glycol (120 g) dissolved in water (about 370 g) isadded and the resulting mixture is stirred until it congeals.

Methods of Treatment

The present compounds are used as therapeutic agents for the treatmentof conditions in mammals that are causally related or attributable toHIV-1 infection. Accordingly, the compounds and pharmaceuticalcompositions of this invention find use as therapeutics for preventingand/or treating diseases causally related or attributable to HIV-1infection, including AIDS, in mammals. In a particular embodiment, themammal is a human.

In a method of treatment aspect, this invention provides a method oftreating a mammal susceptible to or afflicted with a conditionassociated with an HIV-1 infection, which method comprises administeringan effective amount of one or more of the pharmaceutical compositionsjust described.

As a further aspect of the invention there is provided the presentcompounds for use as a pharmaceutical especially in the treatment orprevention of the aforementioned conditions and diseases. Also providedherein is the use of the present compounds in the manufacture of amedicament for the treatment or prevention of one of the aforementionedconditions and diseases.

Injection dose levels range from about 0.1 mg/kg/hour to at least 10mg/kg/hour, all for from about 1 to about 120 hours and especially 24 to96 hours. A preloading bolus of from about 0.1 mg/kg to about 10 mg/kgor more may also be administered to achieve adequate steady statelevels. The maximum total dose is not expected to exceed about 2 g/dayfor a 40 to 80 kg human patient.

For the prevention and/or treatment of long-term conditions, such as,e.g., AIDS and AIDS-related diseases, the regimen for treatment usuallystretches over many months or years, so oral dosing is preferred forpatient convenience and tolerance. With oral dosing, one to five andespecially two to four and typically three oral doses per day arerepresentative regimens. Using these dosing patterns, each dose providesfrom about 0.01 to about 20 mg/kg of the compound of the invention, withpreferred doses each providing from about 0.1 to about 10 mg/kg andespecially about 1 to about 5 mg/kg.

Transdermal doses are generally selected to provide similar or lowerblood levels than are achieved using injection doses. Modes ofadministration suitable for mucosal sites are also envisioned herein andinclude without limitation: intra-anal swabs, enemas, intranasal sprays,and aerosolized or vaporized compounds and/or compositions for deliveryto the lung mucosa. One of skill in the art would choose an appropriatedelivery mode/s based on a variety of parameters, including the organ ortissue site in a patient with a disease or condition that is mostseverely affected by the disease or condition.

The compounds of this invention can be administered as the sole activeagent or they can be administered in combination with other agents,including other compounds that demonstrate the same or a similartherapeutic activity and are determined to safe and efficacious for suchcombined administration.

As described herein, mitochondrial inhibitors or agents identified usingscreening methods of the present invention may be used alone or incombination with other therapeutic agents used for treating subjectsinfected with HIV-1. Accordingly, one or more mitochondrial inhibitorsor agents identified using screening methods of the present inventionmay be administered alone or in conjunction with one or more othertherapeutic agents used for treating subjects infected with HIV-1. Suchtherapeutic agents include: nucleoside analogue reverse transcriptaseinhibitors and either of protease inhibitors or non-nucleoside reversetranscriptase inhibitors, HIV-1 entry or fusion inhibitors, andintegrase inhibitors. For such a combined therapeutic approach, one ormore mitochondrial inhibitors or agents identified using screeningmethods of the present invention may be administered to patient in needthereof concomitantly with one or more other therapeutic agents used fortreating subjects infected with HIV-1, such as NRTIs, proteaseinhibitors, and/or NNRTIs, or may be administered in a therapeuticregimen at appropriately space intervals to achieve optimal therapeuticefficacy.

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLES Materials and Methods

Cell lines and viruses. HIV-1 infected H9 cells, MT2 target cells, andHIV-IIIB were obtained through the AIDS Research and Reference Program,Division of AIDS, National Institute of Allergy and Infectious Diseases,National Institutes of Health. HIV-IIIB and HIV-IIIB infected H9 cellswere originally from R. Gallo [11]. MT2 cells were originally from D.Richman [12].

Cell labeling and co-culture. HIV-1 infected H9 cells were fluorescentlylabeled with MitoTracker Red CMXRos to stain mitochondria red, and withHoechst 33342 to stain DNA blue according to the manufacturer'sprotocols (Molecular Probes). Uninfected MT2 target cells were labeledgreen by the expression of green fluorescent protein (GFP-MT2). Cellswere co-cultured and imaged on a glass bottom 96-well plate (MatTek,Ashland, Mass.) in RPMI 1640 medium containing 10% heat-inactivatedfetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/mlstreptomycin (complete medium). Labeled HIV-1 infected H9 cells werewashed 3 times with complete medium to remove any residual free virusand free dye. Washed cells were suspended in complete medium at adensity of 5×10⁵ cells/ml. 100 μl of this cell suspension, containing50,000 cells, were plated into each well. 100,000 GFP-MT2 target cellsin 100 μl were then added to each well, to give a ratio of 1:2 ofinfected to uninfected target cells.

Live-cell real-time imaging. Live-cell real-time images and time lapsemovies were collected at 37° C., 5% CO₂ using a microplate incubator,with precision temperature, humidity and CO₂ controls, mounted on themicroscope stage of a Leica inverted microscope equipped with anautomatic power stage and an AF6000 camera. Phase contrast, blue(Hoechst 33342), green (GFP), and red (MitoTracker) channels were usedfor imaging. Images were collected at 0.5 min or 5 min intervals for18-24 hours. Image analysis was performed using LCSAF software.

Syncytia formation assay. Acute HIV infection of the target cells as aresult of cell-to-cell transmission of the virus was measured bysyncytia formation assay [13]. Focal syncytium formation was scoredunder an inverted microscope.

HIV-1 p24 assay. Viral replication was assayed by HIV-1 core protein p24expression in the co-culture, using commercial ELISA kit (Coulter,Hialeah, Fla.) as described previously [13].

Data analysis and statistics. For quantitation of syncytial formationand HIV p24 production, data are means ± standard deviations of fourindependent experiments. Experiments were carried out in triplicates ineach run. For live-cell real-time imaging and movies, independentexperiments were repeated at least four times and each time intriplicates.

Results

Mitochondria of HIV Infected Cells Enter Uninfected Target Cells UponContact

FIG. 1 shows that upon contact, mitochondria from an infected H9 cell,stained with MitoTracker Red, enter an uninfected GFP-MT2 target cell.Mitochondrial entry can be followed by location and intensity ofstaining. In the blue-red-green overlay (FIG. 1A), mitochondria enteringthe target cell are seen as yellow spots progressing from thecell-to-cell contact zone to the interior of the target cell. As moremitochondria enter the target cell, the intensity of the yellow in thegreen target cell increases with time. In the absence of the greenoverlay (FIG. 1B), the mitochondria from the HIV-1 infected H9 cell canbe explicitly seen as red stained structures inside the target cell.

Mitochondria of HIV Infected Cells Carry the Virus into the TargetCells; Viral Infection Induces VS and Syncytia Formation and Target CellDepletion

FIG. 2 shows one field of the co-culture at hour 1 (FIG. 2A) and hour 20(FIG. 2B) after mixing in Movie 1 (video 1). At hour 1, variousinfectious cell centers (ICC) are formed, consisting of target cells(green), HIV infected H9 cells (red and blue) and partially infectedtarget cells (red in green appearing as orange and yellow). These ICCs,including VS, nanotubes, and filopodia, are seen around a large centralsyncytium, 125×115 μm. By hour 20, most of the target cells have beendepleted, and new satellite syncytia have formed around the originalcentral syncytium. MitoTracker-stained red mitochondria, both within thesyncytium and free in culture, are seen.

Mitochondria Can Be Transported at a Distance By Nanotubes

Closer examination of one region of interest, outlined by the dottedlines in FIG. 2A, shows the presence of a 25×5 μm nanotube (NT) linkingthe ICC to the syncytium. Detailed analysis of time lapse images every 5minutes shows that discrete groups of MitoTracker-stained mitochondria(white arrows) migrate along the nanotube from the infected cells intothe syncytium at a rate of about 0.3 μm per minute. Selected frames areshown in FIG. 2C. As the mitochondria reach the syncytium, the size andintensity of MitoTracker staining at the surface and interior of thesyncytium increase.

Mitochondria Can Be Transported at a Distance in Groups As an Array

Mitochondria can also be transmitted from HIV infected cells touninfected cells as a mitochondrial array (MA). This is a new mode ofmitochondrial release and movement that we observed in the co-culture.FIG. 2D shows the same field shown in FIG. 2C at hour 11. An array ofcell-free mitochondria stained with MitoTracker Red is seen to emergefrom one of the infected cells (blue arrow). Over a 30 minute period,this mitochondrial array changed direction and navigated toward thesyncytium at a rate of about 0.3 μm per minute (video 1 and FIG. 2D). Weobserved this type of free mitochondrial array many times in theco-cultures.

Cell-Free Mitochondria Purified from HIV Infected Cells Are Infectious

Because mitochondrial transfer from infected cells to uninfected targetcells was observed concurrent with viral spread, the present inventorstested whether highly purified cell-free mitochondria from HIV-infectedcells are infectious. To this end, mitochondria from HIV-infected H9cells were isolated and purified to remove cellular and viralcontaminants, including free virus. Their infectivity was evaluated byadding them to uninfected GFP-MT2 target cells. In the experiment shown,the only intact cells present are uninfected (green) GFP-MT2 targetcells. HIV-infected cells were stained with MitoTracker Red and Hoechstprior to mitochondrial isolation, so as to visualize mitochondriaderived therefrom in co-cultures. Live-cell real-time images are shownin FIG. 3. The results presented herein demonstrate that highly purifiedcell-free mitochondria from HIV-1 infected H9 cells are infectious. Theyharbor the virus and carry the virus into the target cells, infect them,and promote HIV-induced cytopathologic effects on the target cells. Thepresent inventors confirmed viral infection and replication by syncytiaformation and HIV antigen p24 production (FIG. 4).

Mitochondria from HIV-Infected Cells Invade Target Cells; ViralInfection and Replication Promote Target Cell Depletion

FIG. 3A represents a typical field of GFP-labeled MT2 target cells inthe presence of cell-free mitochondria purified from HIV-infected H9cells, one hour after mixing. At this point, green target cells are allclustered with infectious mitochondria into ICCs. FIG. 3B represents thesame field at hour 20. By this time, the entire field of target cellshas been infected, most of them are depleted, and release of freemitochondria can be seen. These second generation cell-free mitochondriaare as infectious as the original mitochondria. To examine the mechanismof cell-free mitochondria in HIV transmission, frame-by-frame analysiswas performed. As seen in FIG. 3C, entry of free mitochondria into aGFP-MT2 target cell results in accumulation of mitochondria (coloredyellow in the red-green-blue overlay) near the site of entry. Viralreplication promotes depletion of the target cell and diminishes its GFPexpression. Finally, at hour 5 and 30 minutes, GFP expression is nolonger visible, but the now infected cell is seen with red mitochondriaand blue nuclear DNA.

Mitochondrial Inhibitors Block HIV Transmission

To verify that functional mitochondria are essential for cell-to-celltransmission of the virus, we pre-treated HIV-1 infected H9 cells withmitochondrial inhibitors including carbonyl cyanidem-chlorophenylhydrazone (CCCP, 10 μM), antimycin A (2.4 μM), andoligomycin (2.4 μM) for 3 hours [14]. These concentrations gave maximuminhibition of the targeted metabolic step and caused no cell death.Cells were washed three times to remove free drug, then suspended incomplete medium, and labeled with MitoTracker Red and Hoechst 33342. Thecells were then co-cultured with GFP-MT2 target cells, and cell-to-celltransmission of HIV-1 was analyzed. Mitochondrial inhibitors reducedcell-to-cell spread of HIV-1 and decreased VS and syncytia formation(FIG. 4C-3).

Confirmation of Mitochondria as HIV Reservoirs and Carriers

To confirm that mitochondria from HIV infected cells serve as viralreservoirs and carry the virus into the uninfected target cells, thepresent inventors quantitated viral infection and replication in theco-cultures by syncytia formation and viral antigen p24 production.These results are seen in FIG. 4. This was achieved using co-cultures ofuninfected MT2 target cells with 1) uninfected H9 cells as controls, 2)cell-free mitochondria purified from uninfected H9 cells, 3)HIV-infected H9 cells, 4) cell-free mitochondria purified from infectedH9 cells, and 5) HIV-infected H9 cells treated with the mitochondrialinhibitor CCCP. As shown in FIGS. 4A and 4B, syncytial formation and p24production were detected in co-cultures of MT2 cells with HIV-infectedH9 cells, as well as with cell-free mitochondria purified fromHIV-infected H9 cells. No syncytia were found with uninfected H9 cells,or with purified cell-free mitochondria from uninfected cells, nor wasthere p24 production. Treatment of HIV-infected H9 cells with CCCPreduced syncytia formation and p24 expression by about 95%. FIG. 4Ccontains fluorescent images showing syncytia formation in co-cultures ofGFP-MT2 target cells with HIV-1 infected H9 cells (FIG. 4C-1) but notwith uninfected H9 cells (FIG. 4C-2). CCCP-treatment of HIV-1 infectedH9 cells inhibits syncytia formation (FIG. 4C-3) while cell-freemitochondria from HIV-1 infected H9 cells promote syncytia formation(FIG. 4C-4).

Discussion

The present inventors used live-cell real-time imaging to followmitochondria stained with MitoTracker Red CMXRos in HIV-1 infected H9cells, in co-culture with uninfected GFP-MT2 target cells. This dye isretained by active mitochondria, and depends on the presence of themitochondrial membrane potential. As described herein, the presentinventors discovered that host mitochondria act as viral reservoirs, andcarry the virus into the target cells, facilitating viral transmission.Furthermore, purified cell-free mitochondria from HIV-1 infected cellsare capable of infecting target cells and forming ICCs consisting of VSand syncytia. In agreement with these findings, pre-treatment ofinfected cells with mitochondrial inhibitors reduced viral transmission.The present inventors confirmed viral transmission, target cellinfection and viral replication by syncytial formation and HIV coreprotein p24 production. Co-cultures of uninfected H9 cells, similarlystained with Hoechst and Mito-Tracker Red, with uninfected GFP-MT2target cells failed to show ICC and syncytial formation, nor did theyshow viral replication or HIV-p24 production.

Overall, results presented herein show that mitochondria of HIV-infectedcells play indispensable roles in cell-to-cell transmission of HIV-1.They suggest that mitochondria act as reservoirs and carriers for HIV-1.How the AIDS virus acts on the host mitochondria and where it hides inthe mitochondria are fascinating questions. The size of HIV-1 is about0.1 μm and that of a mitochondrion is between 1 to 10 μm, so thereshould be ample space for the virus. The number of mitochondria per cellvaries with cell type and metabolic state, and could spike to 1000-2000.Mitochondria are dynamic organelles that undergo rapid cycles of fusionand fission to maintain cellular physiology and function. All of thesemay be useful to the virus for rapid dissemination, in addition toshielding the virus from cellular and immunological barriers.

Membrane fusion is a fundamental process in human life in health and indisease. Transport vesicles fuse with the organelles of the secretorypathway, gametes fuse during fertilization, and enveloped viruses fusewith receptors of the target cells during entry. The fusion mechanismfor these diverse events may be different, but they may operate by somecommon strategies. How HIV interacts with the host mitochondria is animportant and as yet unstudied area. Does the viral fusion protein gp41[15] interact with the mitochondria fusion proteins [16]? Mitochondriaare double membrane-bound organelles. Are both membranes involved in thefusion with the virus?

Results presented herein demonstrate for the first time the essentialrole of mitochondria in cell-to-cell transmission of HIV-1, and revealthat mitochondria and mitochondrial comprising HIV-1 can be transferredbetween cells.

REFERENCES

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This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrate and not restrictive, the scope of the invention beingindicated by the appended claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

What is claimed is:
 1. A method for screening to identify a compound fortreating a subject infected with human immunodeficiency virus type 1(HIV-1), the method comprising contacting HIV-1 infected cells andtarget cells with a candidate compound, wherein the target cells aresusceptible to HIV-1 infection; and measuring cell-to-cell transmissionof HIV-1 in the presence and absence of the candidate compound, whereina reduction in cell-to-cell transmission of HIV-1 in the presence of thecandidate compound relative to the absence of the candidate compoundindicates that the candidate compound or agent is a therapeutic agentfor treating the subject infected with HIV-1.
 2. The method of claim 1,wherein the candidate compound is a mitochondrial inhibitor.
 3. Themethod of claim 2, wherein the candidate compound is a mitochondrialinhibitor and a protease inhibitor.
 4. The method of claim 1, whereinthe candidate compound is a modulator of cytoskeletal processes.
 5. Themethod of claim 4, wherein the cytoskeletal processes relate to actin ormicrotubule polymerization or depolymerization.
 6. The method of claim1, wherein cell-to-cell transmission of HIV-1 is measured by trackingmitochondrial movement from the HIV-1 infected cells to the targetcells.
 7. The method of claim 6, further comprising measuringvirological synapse and syncytia formation.
 8. A method for screening toidentify a compound that inhibits human immunodeficiency virus type 1(HIV-1) uptake by mitochondria, the method comprising contacting HIV-1infected cells and target cells with a candidate compound, wherein thetarget cells are susceptible to HIV-1 infection; and measuring uptake ofHIV-1 by mitochondria in the target cells in the presence and absence ofthe candidate compound, wherein a reduction in uptake of HIV-1 bymitochondria in the target cells in the presence of the candidatecompound relative to the absence of the candidate compound indicatesthat the candidate compound is an inhibitor of HIV-1 uptake by themitochondria.
 9. The method of claim 8, wherein the candidate compoundinhibits viral fusion.
 10. The method of claim 9, wherein the candidatecompound is enfuvirtide (Fuzeon) or maraviroc (Selzentry).
 11. Themethod of claim 8, wherein the candidate compound inhibits activity ofviral fusion protein gp41.
 12. The method of claim 8, wherein thecandidate compound inhibits activity of mitochondrial fusion proteins.13. The method of claim 8, wherein measuring uptake of HIV-1 bymitochondria in the target cells is determined by isolating mitochondriafrom the target cells following contact with HIV-1 infected cells in thepresence or absence of the candidate compound and determining andcomparing infectivity of mitochondria isolated from target cellscontacted with the candidate compound to infectivity of mitochondriaisolated from target cells not contacted with the candidate compound,wherein decreased infectivity in the mitochondria isolated from targetcells contacted with the candidate compound indicates that the candidatecompound is an inhibitor of HIV-1 uptake by the mitochondria.
 14. Amethod for treating a subject infected with human immunodeficiency virustype 1 (HIV-1), the method comprising administering to the subject amitochondrial inhibitor in a therapeutically effective amount, whereinthe therapeutically effective amount is sufficient to reduce or inhibitcell-to-cell mediated transmission of HIV-1, thereby treating thesubject infected HIV-1.
 15. The method of claim 14, wherein themitochondrial inhibitor is ME-344, carbonyl cyanide m-chlorophenylhydrazone (CCCP), antimycin A, or oligomycin.
 16. The method of claim14, wherein the mitochondrial inhibitor is also a proteosome inhibitor.17. The method of claim 16, wherein the mitochondrial and proteosomeinhibitor is bortezomib.
 19. The method of claim 14, further comprisingtreating the subject with a nucleoside analogue reverse transcriptaseinhibitor and either of a protease inhibitor or a non-nucleoside reversetranscriptase inhibitor.